Confocal microscopes are used routinely for viewing internal details of semi-transparent microscopic bodies, especially in biological applications, often employing fluorescence illumination. The essential feature of such a microscope is the illumination of the sample by light focused through a pinhole, combined with observation of the returned light through the same pinhole, the result being that the detected light relates substantially to the specific image plane of the pinhole within the sample, rather than to planes above or below. This permits accurate depth resolution within the sample. Typically, a laser is used to provide a very tightly focused intense beam at the pinhole.
As described above, such a system gives information about only one point in the sample. However, the principle can be extended by two alternative and quite distinct approaches to give an extended image of the sample. In the first method, called ‘scanning spot’ the pinhole is scanned optically over the region of interest and the returned intensity is recorded in order to reconstruct an image of the sample. In the second method, many pinholes are illuminated in parallel to give simultaneous information across the region of interest. One such configuration is the ‘Nipkow disk’ in which the pinholes are set into a disk which is then spun to give multiple scanned coverage of the region. This approach lends itself particularly well to the high speed imaging of live cells, a subject of considerable biological interest currently. Nevertheless, the present invention is relevant to all forms of confocal scanning but most particularly to those employing multiple pinholes.
Some known analysis techniques involve delivery of a powerful beam of light to a selected area of the sample for the purpose of modifying the properties of the sample material in the selected area. For example, many of the dyes used in fluorescence microscopy will ‘bleach’ when exposed to very strong light sources.
Bleaching a particular area allows that area to be ‘tagged’—to be distinguished from adjacent regions which might otherwise be indistinguishable, thereby allowing that area to be tracked as it moves and develops. Such “Fluorescence Recovery After Photobleaching” (or FRAP) allows transport mechanisms within a cell to be monitored. Typically, diffusion processes will cause the bleached spot to recover after a period of time determined by the diffusion rate. For low viscosity media, this can be very fast, in the millisecond range.
A second example of targeted illumination is photoactivation. Some dyes will change their fluorescence colour when activated by a very strong light source. Again, this allows a region to be ‘tagged’ and transport mechanisms to be studied.
Scanning spot confocal systems lend themselves quite well to targeted illumination applications. The single scanning spot covers all parts of the sample and so increasing the laser power while the spot is over the region of interest produces targeted illumination. Despite the fact that such an approach is rather slow and inefficient, targeted illumination has been available on scanning spot systems for some years.
However, this is not the case for spinning disk systems where the parallel illumination of the optical arrangement gives no obvious opportunity to increase the light intensity dramatically over a specified region.